Fermentative preparation process for and crystal forms of cytostatics

ABSTRACT

The invention relates to a new process for concentrating epothilones in culture media, a new process for the production of epothilones, a new process for separating epothilones A and B and a new strain obtained by mutagenesis for the production of epothilones, as well as aspects related thereto. New crystal forms of epothilone B are also described.

This application is a divisional of application Ser. No. 09/248,910,filed Feb. 12, 1999 now U.S. Pat. No. 6,194,181.

Fermentative preparation process for and crystal forms of cytostaticsThe invention relates to a new biotechnological preparation process thatcan be used on an industrial scale for the production of epothilones,especially a process for concentrating these compounds in the culturemedium, as well as a new strain for the fermentative preparation ofthese compounds. The invention also relates to new crystal forms ofepothilones, especially epothilone B, obtainable by the new processes,their usage in the production of pharmaceutical preparations, newpharmaceutical preparations comprising these new crystal forms and/orthe use of these compounds in the treatment of proliferative diseasessuch as tumours, or in the production of pharmaceutical formulationswhich are suitable for this treatment.

BACKGROUND TO THE INVENTION

Of the existing cytotoxic active ingredients for treating tumours,Taxol® (Paclitaxel; Bristol-Myers Squibb), a microtubuli-stabilisingagent, plays an important role and has remarkable commercial success.However, Taxol has a number of disadvantages. In particular, its verypoor solubility in water is a problem. It therefore became necessary toadminister Taxol® in a formulation with Cremophor EL® (polyoxyethylatedcastor oil; BASF, Ludwigshafen, Germany). Cremophor EL® has severe sideeffects; for example it causes allergies which in at least one case haveled even to the death of a patient.

Although the Taxan class of microtubuli-stabilising anti-cancer agentshas been commended as “perhaps the most important addition to thepharmaceutical armoury against cancer in the last decade” (see RowinskyE. K., Ann. rev. Med. 48, 353-374 (1997)), and despite the commercialsuccess of Taxol®, these compounds still do not appear to represent areally great breakthrough in the chemotherapy of cancer. Treatment withTaxol® is linked with a series of significant side effects, and a fewprimary classes of compact tumours, namely colon and prostate tumours,respond to this compound only to a small extent (see Rowinsky E. K.,inter alia). In addition, the efficacy of Taxol can be impaired and evencompletely neutralised by acquired resistance mechanisms, especiallythose based on the overexpression of phosphoproteins, which act asefflux pumps for active ingredients, such as “Multidrug Resistance” dueto overexpression of the multidrug transport glycoprotein“P-glycoprotein”.

Epothilones A and B represent a new class of microtubuli-stabilisingcytotoxic active ingredients (see Gerth, K. et al., J. Antibiot. 49,560-3 (1966)) of the formulae:

wherein R signifies hydrogen (epothilone A) or methyl (epothilone B).

These compounds have the following advantages over Taxol®:

a) They have better water-solubility and are thus more easily accessiblefor formulations.

b) It has been reported that, in cell culture experiments, they are alsoactive against the proliferation of cells, which, owing to the activityof the P-glycoprotein efflux pump making them “multidrug resistant”,show resistance to treatment with other chemotherapy agents includingTaxol® (see Bolag, D. M., et al., “Epothilones, a new class ofmicrotubuli-stabilizing agents with a Taxol-like mechanism of action”,Cancer Research 55, 2325-33 (1995)). And

c) it could be shown that they are still very effective in vitro againsta Taxol®-resistant ovarian carcinoma cell line with modified β-tubulin(see Kowalski, R. J., et al., J. Biol. Chem. 272(4), 2534-2541 (1997)).

Pharmaceutical application of the epothilones, for example for tumourtreatment, is possible in an analogous manner to that described forTaxol, see for example U.S. Pat. No. 5,641,803; U.S. Pat. No. 5,496,804;U.S. Pat. No. 5,565,478).

In order to be able to use the epothilones on a larger scale forpharmaceutical purposes, however, it is necessary to obtain appropriateamounts of these compounds.

Until now, the extraction of natural substances by means ofmyxobacteria, especially the epothilones from the cell strain Sorangiumcellulosum Soce90 (deposited under no. 6773 at the German Collection ofMicroorganisms, see WO 93/10121) was described in literature. In orderto obtain a satisfactory concentration of the natural substances,especially the epothilones, in the culture medium for the subsequentextraction, previously an adsorbate resin based on polystyrene wasalways added, for example Amberlite XAD-1180 (Rohm & Haas, Frankfurt,Germany).

However, the disadvantage of this process is that, on a large scale, itleads to an abundance of problems. Valves are impaired by the globulesof resin, pipes can block, and apparatus may be subject to greater weardue to mechanical friction. The globules of resin are porous andtherefore have a large inner surface area (about 825 m²/gram resin).Sterilisation becomes a problem, as air enclosed in the resin is notautoclaved. Thus, the process cannot be practicably carried out on alarge scale using resin addition.

On the other hand, without adding resin globules, a satisfactoryconcentration of epothilones cannot be achieved in the culture medium.

Surprisingly, the requirements for finding a way out of this dilemmahave now been found, enabling a satisfactory concentration of naturalsubstances to be obtained from microorganisms, in particularmyxobacteria, which produce epothilones such as epothilone A or B, inparticular a concentration of epothilones A and B, in the culturemedium, without the addition of resins, and thus enabling production ofthese compounds, especially epothilones to be carried out on a technicaland industrial scale without the above-mentioned disadvantages.

DETAILED DESCRIPTION OF THE INVENTION

One aspect of the invention relates to a process for concentratingepothilones, especially epothilone A and/or B, in particular epothiloneB, in a culture medium, in order to produce these compounds on abiotechnological scale, the process comprising microorganisms whichproduce these compounds, especially myxobacteria (as producers ofnatural substances), whereby a complex-forming components which issoluble in the culture medium is added to the medium.

A further aspect relates to the corresponding culture medium, whichcomprises a corresponding complex-forming component and microorganisms,especially myxobacteria, in particular of the genus Sorangium, which aresuitable for producing epothilones, especially epothilone A and/or B.

A further aspect of the invention relates to a process for theproduction of epothilones, especially epothilone A and/or B, especiallythe two pure compounds, in particular epothilone B, which ischaracterised in that the epothilones are obtained by working up aculture medium for the biotechnological preparation of these compounds,which comprises as producers of natural substances microorganisms,especially myxobacteria, that produce these compounds, and to which acomplex-forming component that is soluble in the culture medium isadded, and the subsequent purification and, if desired, separation ofthe epothilones, for example epothilone A and B.

A fourth aspect of the invention relates to a method of separatingepothilones, especially epothilones A and B from one another, which ischaracterised by chromatography on a reversed-phase column with aneluant comprising a lower alkyl cyanide.

A further aspect of the invention relates to a strain of Sorangiumcellulosum obtained by mutagenesis, which under otherwise identicalconditions, produces more epothilones than Sorangium cellulosum Soce90.

A further aspect also relates to new crystal forms of epothilone B.

The general terms used hereinabove and hereinbelow preferably have themeanings given hereinbelow:

Where reference is made hereinabove and hereinbelow to documents, theseare incorporated insofar as is necessary.

The prefix “lower” always indicates that the correspondingly namedradical contains preferably up to a maximum of 7 carbon atoms, inparticular up to 4 carbon atoms, and is branched or unbranched. Loweralkyl may be for example unbranched or branched once or more, and ise.g. methyl, ethyl, propyl such as isopropyl or n-propyl, butyl such asisobutyl, sec.-butyl, tert.-butyl or n-butyl, or also pentyl such asamyl or n-pentyl.

A culture medium for the biotechnological preparation of epothiloneswhich contains microorganisms that produce these compounds, especiallymyxobacteria, as producers of natural substances, is primarily a mediumwhich comprises a corresponding (naturally occurring or also obtainableby cultivation or in particular by genetic modification) microorganism,especially a myxobacterial strain which is in a position to producethese compounds, in particular a myxobacterium of the genus Sorangium,preferably (in particular for epothilone production) a microorganism ofthe type Sorangium cellulosum Soce90 (see WO 93/10121), or a biomaterialderived therefrom or from parts of this myxobacterium, especially acorrespondingly derived strain, in particular the strain having thereference BCE33/10, in particular the strain having the reference BCE63/114 or mutants thereof, and in addition, together with water,preferably other conventional and appropriate constituents of culturemedia, such as biopolymers, sugar, amino acids, salts, nucleic acids,vitamins, antibiotics, semiochemicals, growth media, extracts frombiomaterials such as yeast or other cell extracts, soy meal, starch suchas potato starch and/or trace elements, for example iron ions incomplex-bound form, or suitable combinations of all or some of theseconstituents and/or also analogous additions. The corresponding culturemedia are known to the person skilled in the art or may be produced byknown processes (see e.g. the culture media in the examples of thepresent disclosure, or in WO 93/10121).

One preferred myxobacterium is a strain selected by UV mutagenesis andselection for increased formation of epothilone A and/or B overSorangium cellulosum Soce90, which is deposited in the DSM under no.6773, especially the mutant BCE33/10, which was deposited under thenumber DSM 11999 on Feb. 9th, 1998 at the German Collection ofMicroorganisms and Cell Cultures (DSMZ, Braunschweig, Germany), and mostpreferably the mutant having the reference BCE 63/114, which wasdeposited under number DSM 12539 on Nov. 27th, 1998 at the GermanCollection of Microorganisms and Cell Cultures (DSMZ).

Strain culture and morphological description of strain BCE 33/10 and ofstrain BCE 63/11 4: The strain can grow on cellulose as the sole sourceof carbon and energy with potassium nitrate as the sole source ofnitrogen, e.g. on filter paper over ST21 mineral salt agar (0.1% KNO₃;0.1% MgSO₄×7 H₂O; 0.1% CaCl₂×2 H₂O; 0.1% K₂HPO₄; 0.0% MnSO₄×7 H₂O; 0.02%FeCl₃; 0.002% yeast extract; standard trace element solution; 1% agar).On this medium, dark reddish-brown to blackish-brown fruiting bodies areformed. They consist of small sporangioles (ca. 15 to 30 μm diameter)and exist in dense heaps and packs of varying size.

The vegetative bacilli have the shape typical of Sorangium (relativelycompact, under the phase contrast microscope dark, cylindrical bacilliwith broad rounded ends, on average 3 to 6 μm long and 1 μm thick).

Epothilones are primarily epothilone A and/or B, but also otherepothilones, for example epothilones C and D named in InternationalApplication WO 97/19086 and WO 98/22461, epothilones E and F named in WO98/22461, and further epothilones obtainable from correspondingmicroorganisms.

A water-soluble complex-forming component is primarily a water-solubleoligo- or polypeptide derivative or in particular an oligo- orpolysaccharide derivative of cyclic or helical structure, which forms anintramolecular cavity, which because of its sufficiently hydrophobicproperties is in a position to bind epothilones, especially epothilone Aand/or epothilone B. A water-soluble complex-forming component that isespecially preferred is one that is selected from cyclodextrins or (inparticular) cyclodextrin derivatives, or mixtures thereof.

Cyclodextrins are cyclic (α-1,4)-linked oligosaccharides ofα-D-glucopyranose with a relatively hydrophobic central cavity and ahydrophilic external surface area.

The following are distinguished in particular (the figures inparenthesis give the number of glucose units per molecule):α-cyclodextrin (6), β-cyclodextrin (7), γ-cyclodextrin (8),δ-cyclodextrin (9), ε-cyclodextrin (10), ζ-cyclodextrin (11),η-cyclodextrin (12), and θ-cyclodextrin (13). Especially preferred areδ-cyclodextrin and in particular α-cyclodextrin, β-cyclodextrin orγ-cyclodextrin, or mixtures thereof.

Cyclodextrin derivatives are primarily derivatives of theabove-mentioned cyclodextrins, especially of α-cyclodextrin,β-cyclodextrin or γ-cyclodextrin, primarily those in which one or moreup to all of the hydroxy groups (3 per glucose radical) are etherifiedor esterified. Ethers are primarily alkyl ethers, especially loweralkyl, such as methyl or ethyl ether, also propyl or butyl ether; thearyl-hydroxyalkyl ethers, such as phenyl-hydroxy-lower-alkyl, especiallyphenyl-hydroxyethyl ether; the hydroxyalkyl ethers, in particularhydroxy-lower-alkyl ethers, especially 2-hydroxyethyl, hydroxypropylsuch as 2-hydroxypropyl or hydroxybutyl such as 2-hydroxybutyl ether;the carboxyalkyl ethers, in particular carboxy-lower-alkyl ethers,especially carboxymethyl or carboxyethyl ether; derivatised carboxyalkylethers, in particular derivatised carboxy-lower-alkyl ether in which thederivatised carboxy is etherified or amidated carboxy (primarilyaminocarbonyl, mono- or di-lower-alkyl-aminocarbonyl, morpholino-,piperidino-, pyrrolidino- or piperazino-carbonyl, or alkyloxycarbonyl),in particular lower alkoxycarbonyl-lower-alkyl ether, for examplemethyloxycarbonylpropyl ether or ethyloxycarbonylpropyl ether; thesulfoalkyl ethers, in particular sulfo-lower-alkyl ethers, especiallysulfobutyl ether; cyclodextrins in which one or more OH groups areetherified with a radical of formula

—O-[alk-O—]_(n)—H

wherein alk is alkyl, especially lower alkyl, and n is a whole numberfrom 2 to 12, especially 2 to 5, in particular 2 or 3; cyclodextrins inwhich one or more OH groups are etherified with a radical of formula

wherein R′ is hydrogen, hydroxy, —O-(alk-O)_(z)—H, —O-(alk(-R)—O—)_(p)—Hor —O-(alk(-R)—O—)_(q)-alk-CO—Y; alk in all cases is alkyl, especiallylower alkyl; m, n, p, q and z are a whole number from 1 to 12,preferably 1 to 5, in particular 1 to 3; and Y is OR₁ or NR₂R₃, whereinR₁, R₂ and R₃ independently of one another, are hydrogen or lower alkyl,or R₂ and R₃ combined together with the linking nitrogen signifymorpholino, piperidino, pyrrolidino or piperazine; or branchedcyclodextrins, in which etherifications or acetals with other sugars arepresent, especially glucosyl-, diglucosyl-(G₂-β-cyclodextrin), maltosyl-or dimaltosyl-cyclodextrin, or N-acetylglucosaminyl-, glucosaminyl-,N-acetylgalactosaminyl- or galactosaminyl-cyclodextrin.

Esters are primarily alkanoyl esters, in particular lower alkanoylesters, such as acetyl esters of cyclodextrins.

It is also possible to have cyclodextrins in which two or more differentsaid ether and ester groups are present at the same time.

Mixtures of two or more of the said cyclodextrins and/or cyclodextrinderivatives may also exist.

Preference is given in particular to α-, β- or γ-cyclodextrins or thelower alkyl ethers thereof, such as methyl-β-cyclodextrin or inparticular 2,6-di-O-methyl-β-cyclodextrin, or in particular the hydroxylower alkyl ethers thereof, such as 2-hydroxypropyl-α-,2-hydroxypropyl-β- or 2-hydroxypropyl-γ-cyclodextrin.

The cyclodextrins or cyclodextrin derivatives are added to the culturemedium preferably in a concentration of 0.02 to 10, preferably 0.05 to5, especially 0.1 to 4, for example 0.1 to 2 percent by weight (w/v).

Cyclodextrins or cyclodextrin derivatives are known or may be producedby known processes (see for example U.S. Pat. No. 3,459,731; U.S. Pat.No. 4,383,992; U.S. Pat. No. 4,535,152; U.S. Pat. No. 4,659,696; EP 0094 157; EP 0 149 197; EP 0 197 571; EP 0 300 526; EP 0 320 032; EP 0499 322; EP 0 503 710; EP 0 818 469; WO 90/12035; WO 91/11200; WO93/19061; WO 95/08993; WO 96/14090; GB 2,189,245; DE 3,118,218; DE3,317,064 and the references mentioned therein, which also refer to thesynthesis of cyclodextrins or cyclodextrin derivatives, or also: T.Loftsson and M. E. Brewster (1996): Pharmaceutical Applications ofCyclodextrins: Drug Solubilization and Stabilisation: Journal ofPharmaceutical Science 85 (10):1017-1025; R. A. Rajewski and V. J.Stella(1996): Pharmaceutical Applications of Cyclodextrins: In Vivo DrugDelivery: Journal of Pharmaceutical Science 85 (11): 1142-1169).

In the following description of the working up, “epothilone” isunderstood to be an epothilone which is obtainable from thecorresponding microorganism, preferably epothilone C, D, E, F orespecially A or in particular epothilone B. If not otherwise stated,where “epothilones” are mentioned, this is intended to be a general termwhich includes individual epothilones or mixtures.

Working up of the epothilones is effected by conventional methods; firstof all, by separating a culture into the liquid phase (centrifugate orfiltrate) and solid phase (cells) by means of filtration orcentrifugation (tubular centrifuge or separator). The (main) part of theepothilones found in the centrifugate or in the filtrate is thendirectly mixed with a synthetic resin, for example a resin based onpolystyrene as matrix (hereinafter referred to also simply aspolystyrene resin), such as Amberlite XAD-16 [Rohm & Haas Germany GmbH,Frankfurt] or Diaion HP-20 [Resindion S. R. L., Mitsubishi Chemical Co.,Milan] (preferably in a ratio of centrifugate: resin volume of ca. 10:1to 100:1, preferably about 50:1). After a period of contact ofpreferably 0.25 to 50 hours, especially 0.8 to 22 hours, the resin isseparated, for example by filtration or centrifugation. If required,after adsorption, the resin is washed with a strongly polar solvent,preferably with water. Desorption of the epothilones is then effectedwith an appropriate solvent, especially with an alcohol, in particularisopropanol. The solvent phase, especially the isopropanol phase, isthen removed from the solvent, preferably by means of prior,simultaneous or subsequent addition of water, in particular in acirculating evaporator, thereby being concentrated if necessary, and theresulting water phase is extracted with a solvent suitable for forming asecond phase, such as an ester, for example a lower alkanol loweralkanoate, typically ethyl acetate or isopropyl acetate. The epothilonesare thereby transferred into the organic phase. Then the organic phaseis concentrated to the extent necessary, preferably to dryness, forexample using a rotary evaporator.

Subsequently, further processing takes place using the following steps,whereby the purification step by means of reversed-phase chromatographywith elution with a nitrile is an inventive step and is thus compulsory,while the other steps are optional:

molecular filtration (gel chromatography), e.g. on a column of materialsuch as Sephadex LH-20 (Pharmacia, Uppsala, Sweden) with an alcohol suchas methanol as eluant;

separation of the epothilones by reversed-phase chromatography afterbeing taken up in a suitable solvent, and elution with a mixture ofnitrile/water (compulsory), preferably characterised in that thechromatography is carried out on a column of material, especially aRP-18 material, which is charged with hydrocarbon chains, such ashydrocarbon chains containing 18 carbon atoms, and an eluant comprisinga nitrile, especially a lower alkyl-nitrile, in particular acetonitrile,is used, in particular a mixture of nitrile/water is used, especially amixture of acetonitrile/water, preferably in a ratio of nitrile to waterof about 1:99 to 99:1, primarily between 1:9 and 9:1, e.g. between 2:8and 7:3, e.g. 3:7 or 4:6.

single or multiple extraction of the residue (especially afterevaporation) in a two-phase system consisting of water and a solventimmiscible with water, preferably an ester, in particular a lower alkyllower alkanoate, such as ethyl acetate or isopropyl acetate;

adsorption chromatography, in particular by adding to a column of silicagel and eluting with an appropriate solvent or solvent mixture,especially a mixture of ester/hydrocarbon, for example lower alkylalkanoate/C₄-C₁₀-alkane, especially ethyl or isopropyl acetate/n-hexane,in which the ratio between the ester and hydrocarbon is preferably inthe range 99:1 to 1:99, preferably 10:1 to 1:10, for example 4:1;

dissolving the residue, which may be obtained after concentration, in anappropriate solvent such as an alcohol, e.g. methanol;

mixing with activated carbon and removal thereof;

recrystallisation, e.g. from appropriate solvents or solvent mixtures,for example consisting of esters, ester/hydrocarbon mixtures oralcohols, especially ethyl or isopropyl acetate:toluene 1:10 to 10:1,preferably 2:3 (epothilone A) or methanol or ethyl acetate (epothiloneB);

whereby between each step being employed, the resulting solutions orsuspensions are concentrated if necessary, and/or liquid and solidcomponents are separated from one another, in particular by filtering orcentrifuging solutions/suspensions. The more precise definitionsmentioned below can be preferably used in the above individual steps.

The further working up and purification is preferably carried out

either by direct separation of the epothilones from one another byreversed-phase chromatography after being taken up in an appropriatesolvent, for example a nitrile/water mixture, especially anacetonitrile/water mixture (ratio of nitrile to water 1:99 to 99:1,preferably 1:9 to 9:1, especially 3:1), if necessary after filtration orcentrifugation, preferably on a silica gel that has been derivatized byhydrocarbon radicals, e.g. a silica gel modified by alkyl radicalscontaining 8 to 20, especially 18, C-atoms, eluting with an eluantcomprising a nitrile, especially a lower alkylnitrile, such asacetonitrile, especially a mixture of the nitrile with water, such as anacetonitrile/water mixture, whereby detection of the interestingfractions is effected in conventional manner, for example by UVdetection or (preferably) by on-line HPLC (HPLC with a very smallcolumn, the analyses taking less than 1 minute, and detection e.g. at250 nm), this enabling a particularly exact separation of the fractionscontaining the desired product to take place; if required, withsubsequent concentration, for example by distillation, to remove thenitrile; if desired, with subsequent single or multiple, for exampledouble, extraction of the residue of evaporation in a two-phase systemconsisting of water and an immiscible solvent, such as ethyl acetate orisopropyl acetate; additional concentration of the organic phase anddissolving of the residue in an appropriate solvent, preferably an estersuch as ethyl acetate or isopropyl acetate, if required, filtration orcentrifugation, if desired adding to a column of silica gel and elutingwith an appropriate solvent or solvent mixture, for example with amixture of ester/hydrocarbon, e.g. lower alkyl alkanoate/C₄-C₁₀-alkane,especially ethyl or isopropyl acetate/n-hexane, in which the ratio ofester to hydrocarbon is preferably in the range 99:1 to 1:99, preferably10:1 to 1:10, e.g. 4:1; subsequent combining of the fractions containingeach desired epothilone, especially epothilone A or epothilone B, andafter removing the solvent, for example by distillation, preferablyconcentrating to dryness; then, dissolving of the residue in anappropriate alcohol, preferably methanol; and if desired, in order toobtain especially high purity, mixing with activated carbon and thenseparating the activated carbon, for example by filtration; and finally,by recrystallisation as described below under variant 2 (for epothiloneB in particular from methanol), separate extraction of the epothilones,especially epothilones A or B. This is the most preferred variant 1, theoutstanding characteristic of which is the surprising direct separationby reversed-phase chromatography of the epothilone-containing mixturedesorbed by the resin, despite all the impurities in the organicextract;

or (variant 2) first of all exclusion chromatography takes place(molecular filtration) e.g. on a column of material such as SephadexLH-20 (Pharmacia, Uppsala, Sweden) with an alcohol such as methanol aseluant, and then subsequent separation of the epothilones present in thepeak fractions obtained, e.g. epothilone A and B, by reversed-phasechromatography as described above for variant 1; if required twice, ifpeak fractions of one epothilone contain those of another, for exampleif those with epothilone A still contain residues of epothilone B; andthen separate recrystallisation of each epothilone from appropriatesolvents o r solvent mixtures, for example from ethyl or isopropylacetate:toluene 1:10 to 10:1, preferably 2:3 (epothilone A) or methanolor ethyl acetate (epothilone B). This is variant 2 of working up andpurification.

With variant 1, highly pure epothilone B may be obtained in a relativelysimpler manner (the purity is preferably greater than 97%, especiallyover 99%).

Variant 1 preferably takes place as follows (whereby preferably theabove-mentioned variants can be used instead of the following generaldefinitions): First of all, a culture is separated into the liquid phase(centrifugate or filtrate) and a solid phase (cells) by means offiltration or centrifugation (tubular centrifuge or separator). The(main) part of the epothilones found in the centrifugate or filtrate isthen directly mixed with a synthetic resin. After a contact period ofpreferably 0.25 to 50 hours, the resin is separated, for example byfiltration or centrifugation. If required, after adsorption, the resinis washed with a strongly polar solvent, preferably with water.Desorption of the epothilones is then effected with an appropriatesolvent, especially with an alcohol, in particular isopropanol. Thesolvent phase, especially isopropanol phase, is then removed from thesolvent, preferably by means of prior, simultaneous or subsequentaddition of water, in particular in a circulating evaporator, therebybeing concentrated if necessary, and the resulting water phase isextracted with a solvent suitable for forming a second phase , such asan ester, for exampl e a lower alkanol lower alkanoate, typically ethylacetate or isopropyl acetate . The epothilones are t hen transferredinto the organic phase. Then the organic phase is concentrated to theextent necessary, preferably to dryness, for example using a rotaryevaporator. By subsequent reversed-phase chromatography on a silica gelderivatized with hydrocarbon atoms, e.g. a silica gel modified by alkylradicals containing 18 C-atoms, and eluting with a mixture of a loweralkylnitrile such as acetonitrile with water, the epothilones aredirectly separated from one another, especially epothilone A andepothilone B; then, concentration takes place by means of distillation,the residue is shaken out once or more, if desired from water with anappropriate solvent that is immiscible with water, preferably an estersuch as isopropyl acetate, then the organic phase is again concentratedand the residue of evaporation is dissolved in an ester such as ethyl orisopropyl acetate, filtered if required, the filtrate added to a columnof silica gel, and eluted with a mixture of ester/hydrocarbon, e.g.ethyl or isopropyl acetate/n-hexane; subsequently, the fractionscontaining the epothilone, especially epothilone A or B, arerespectively combined and, after removing the solvent by distillation,concentrated, preferably to dryness; the residue is then dissolved in anappropriate lower alkanol, preferably methanol, and in order to obtainespecially high purity, mixed with activated carbon and then filtered;finally, the epothilones are extracted by recrystallisation (in the caseof epothilone B preferably from methanol).

Variant 2 is effected preferably as follows: After harvest, a culture isseparated into the liquid phase (centrifugate) and solid phase (cells)by means of centrifugation (tubular centrifuge or separator). The mainpart of the epothilones are found in the centrifugate, which is thendirectly mixed with a polystyrene resin, such as Amberlite XAD-16 [Rohm& Haas Germany GmbH, Frankfurt] or Diaion HP-20 [Resindion S.R.L.,Mitsubishi Chemical Co., Milan] (preferably in a ratio of centrifugate:resin volume of ca. 10:1 to 100:1, preferably about 50:1) and stirred inan agitator. In this step, the epothilones are transferred from thecyclodextrin to the resin. After a period of contact of ca. 1 hour, theresin is separated by centrifugation or filtration. Adsorption of theepothilones onto the resin may also be effected in a chromatographycolumn, by placing the resin in the column and running the centrifugateover the resin. After adsorption, the resin is washed with water.Desorption of the epothilones from the resin is effected withisopropanol. The isopropanol phase is then freed of isopropanolpreferably by the addition of water in particular in a circulatingevaporator, and the resulting water phase is extracted with ethylacetate. The epothilones are thus transferred from the water phase tothe ethyl acetate phase. Then the ethyl acetate extract is concentratedto dryness, for example using a rotary evaporator. An initialconcentration of the epothilones is then achieved by means of molecularfiltration (e.g. Sephadex LH-20 [Pharmacia, Uppsala, Sweden] withmethanol as eluant). The peak fractions from the molecular filtrationcontain epothilone A and B and have a total epothilone content of >10%.Separation of these peak fractions, which contain epothilone A and B ina mixture, into the individual components, then follows by means ofchromatography on a “reversed-phase”, e.g. RP-18 phase (phase which ismodified by alkyl radicals containing 18 carbon atoms per chain), withan appropriate eluant, preferably one containing a nitrile such asacetonitrile (this gives better separation than for example alcoholssuch as methanol). Epothilone A elutes before epothilone B. The peakfractions with epothilone B may still contain small portions ofepothilone A, which can be removed by further separation on RP-18.Finally, the epothilone A fraction is crystallised directly from ethylacetate:toluene=2:3, and the epothilone B fraction from methanol orethyl acetate.

Preferred Embodiment of the Invention

The invention preferably relates to a process for the concentration ofepothilones, especially epothilone A and/or B, in particular epothiloneB, in a culture medium for the biotechnological preparation of this(these) compound(s), which contains a microorganism which is suitablefor this preparation, especially a Sorangium strain, especially of thetype Sorangium cellulosum Soce90, or a mutant arising therefrom, inparticular the strain having reference BCE 33/10, especially the strainhaving reference BCE 63/114, water and other usual appropriateconstituents of culture media, whereby a cyclodextrin or a cyclodextrinderivative, or a mixture of two or more of these compounds is added tothe medium, especially one or more, preferably one or two or morecyclodextrins selected from α-cyclodextrin (6), β-cyclodextrin (7),γ-cyclodextrin (8), δ-cyclodextrin (9), ε-cyclodextrin (10),ζ-cyclodextrin (11), η-cyclodextrin (12), and θ-cyclodextrin (13),especially α-cyclodextrin, β-cyclodextrin or γ-cyclodextrin; orprimarily a cyclodextrin derivative or mixture of cyclodextrinderivatives selected from derivatives of a cyclodextrin, in which one ormore up to all of the hydroxy groups are etherified to an alkyl ether,especially lower alkyl, such as methyl or ethyl ether, also propyl orbutyl ether; an aryl-hydroxyalkyl ether, such asphenyl-hydroxy-lower-alkyl, especially phenyl-hydroxyethyl ether; ahydroxyalkyl ether, in particular hydroxy-lower-alkyl ethers, especially2-hydroxyethyl, hydroxypropyl such as 2-hydroxypropyl or hydroxybutylsuch as 2-hydroxybutyl ether; a carboxyalkyl ether, in particularcarboxy-lower-alkyl ether, especially carboxymethyl or carboxyethylether; a derivatised carboxyalkyl ether, in particular a derivatisedcarboxy-lower-alkyl ether in which the derivatised carboxy isaminocarbonyl, mono- or di-lower-alkyl-aminocarbonyl, morpholino-,piperidino-, pyrrolidino- or piperazino-carbonyl, or alkyloxycarbonyl,in particular lower alkoxycarbonyl, such as preferably loweralkoxycarbonyl-lower-alkyl ether, for example methyloxycarbonylpropylether or ethyloxycarbonylpropyl ether; a sulfoalkyl ether, in particularsulfo-lower-alkyl ether, especially sulfobutyl ether; a cyclodextrin inwhich one or more OH groups are etherified with a radical of formula

—O-[alk-O—]_(n)—H

wherein alk is alkyl, especially lower alkyl, and n is a whole numberfrom 2 to 12, especially 2 to 5, in particular 2 or 3; a cyclodextrin inwhich one or more OH groups are etherified with a radical of formula

wherein R′ is hydrogen, hydroxy, —O-(alk-O)_(z)—H, —O-(Alk(-R)—O—)_(p)—Hor —O-(alk(-R)—O—)_(q)-alk-CO—Y; alk in all cases is alkyl, especiallylower alkyl; m, n, p, q and z are a whole number from 1 to 12,preferably 1 to 5, in particular 1 to 3; and Y is OR₁ or NR₂R₃, whereinR₁, R₂ and R₃ independently of one another, are hydrogen or lower alkyl,or R₂ and R₃ combined together with the linking nitrogen signifymorpholino, piperidino, pyrrolidino or piperazino; or a branchedcyclodextrin, in which etherifications or acetals with other sugars arepresent, and which are selected from glucosyl-,diglucosyl-(G₂-β-cyclodextrin), maltosyl- or dimaltosyl-cyclodextrin, orN-acetylglucosaminyl-, glucosaminyl-, N-acetylgalactosaminyl- andgalactosaminyl-cyclodextrin; or a lower alkanoyl, such as acetyl esterof a cyclodextrin.

Particular preference is given to a process in which the cyclodextrinand/or the cyclodextrin derivative is added to the culture medium in aconcentration of 0.02 to 10, preferably 0.005 to 10, more preferably0.05 to 5, most preferably 0.1 to 4, for example 0.1 to 2, percent byweight (w/v).

Especially preferred is a process according to one of the two previousparagraphs, in which the cyclodextrin derivative is selected from acyclodextrin, especially β-cyclodextrin, and a hydroxy loweralkyl-cyclodextrin, especially 2-hydroxypropyl-α-, -β- or-γ-cyclodextrin; or mixtures of one or more thereof; whereby2-hydroxypropyl-β-cyclodextrin on its own is preferred in particular.

The invention also relates in particular to a culture medium, whichcomprises a cyclodextrin, a cyclodextrin derivative or a mixture of twoor more complex-forming components selected from cyclodextrins andcyclodextrin derivatives, especially a cyclodextrin or cyclodextrinderivative as defined in the third-last paragraph, in particular as inthe second-last paragraph, or a mixture of one or more of thesecompounds, and a microorganism which is suitable for producingepothilones, especially epothilone A and/or B, preferably a strain fromthe genus Sorangium, especially a strain of Sorangium cellulosum, e.g.the strain Soce90 or a mutant arising therefrom, in particular thestrain BCE 33/10, or especially BCE 63/114.

A further aspect of the invention relates to a process for theproduction of epothilone A and/or B, especially the two pure compounds,in particular epothilone B, which is characterised in that theepothilones are separated for example by centrifugation into the solidand the liquid phase (centrifugate) by working up a culture medium forthe biotechnological preparation of these compounds, as described above,to which has been added a complex-forming component which is soluble inthe culture medium, in particular a cyclodextrin, a cyclodextrinderivative or a mixture of two or more cyclodextrins and/or cyclodextrinderivatives; the centrifugate is mixed with a resin, especially apolystyrene resin, or is run through a column filled with such a resin;if necessary, the resin is washed with water; the epothilone(s) is orare desorbed from the resin using a polar solvent, especially analcohol, primarily a lower alkanol such as isopropanol; if necessary,concentrated by means of prior, simultaneous or subsequent addition ofwater; an organic solvent which is immiscible with water, for example anester, such as ethyl acetate, is added, and the epothilone(s) is or aretransferred to the organic phase, for example by agitating or stirring;where necessary, the organic phase is concentrated; the epothilones fromthe organic solution obtained are concentrated through a molecular sievefor compounds of low molecular weight; and then the fractions containingthe epothilones, especially epothilone A and/or B undergo separation bya reversed-phase column, preferably eluting with an eluant containing anitrile, such as acetonitrile (or instead, an eluant containing analcohol, such as methanol); whereby epothilones A and B are extractedseparately, and if desired, can be further concentrated byrecrystallisation.

One preferred aspect of the invention relates to a process for theproduction of epothilone A and/or B, especially the two pure compounds,in particular epothilone B, which is characterised in that theepothilones are separated for example by centrifugation into the solidand the liquid phase (centrifugate) by working up a culture medium forthe biotechnological preparation of these compounds, as described above,to which has been added a complex-forming component which is soluble inthe culture medium, in particular a cyclodextrin, a cyclodextrinderivative or a mixture of two or more cyclodextrins and/or cyclodextrinderivatives; the centrifugate is mixed with a resin, especially apolystyrene resin, or is run through a column filled with such a resin;if necessary, the resin is washed with water; the epothilone(s) is orare desorbed from the resin using a polar solvent, especially analcohol, primarily a lower alkanol such as isopropanol; if necessary,the polar solvent is removed by means of prior, simultaneous orsubsequent addition of water; the resulting water phase is extractedwith a solvent which is suitable for forming a second phase, for examplean ester, such as diethyl ester, if necessary, the organic phase isconcentrated, preferably to dryness; epothilone A and B are separatedfrom one another directly by reversed-phase chromatography, eluting withan eluant containing a nitrile, especially a lower alkylnitrile, such asacetonitrile, whereby detection is effected in the usual manner, forexample by UV detection or preferably by on-line HPLC (HPLC with a verysmall column, the analyses taking less than 1 minute, and detection e.g.at 250 nm); subsequent concentration, for example by distillation; ifdesired, the residue is treated from an aqueous solution once or more(for example twice) by extraction with a solvent which is immisciblewith water, such as an ester; dissolved in an appropriate solvent,preferably an ester such as ethyl or isopropyl acetate, filtered ifnecessary, added to a column of silica gel and eluted with anappropriate solvent or solvent mixture, for example with anester/hydrocarbon mixture; and subsequently, the fractions containingeither epothilone A or especially B are separately combined and, afterremoving the solvent, for example by distillation, concentratedpreferably to dryness; then the residue is dissolved in an appropriatealcohol, preferably methanol, then if desired, in order to obtainespecially high purity, treated with activated carbon and then filtered;and finally epothilone A or B is obtained by recrystallisation (in thecase of epothilone B, particularly from methanol).

A further preferred aspect of the invention relates to a method ofseparating epothilones, especially epothilones A and B from one another,which is characterised by chromatography on a reversed-phase column withan eluant containing a lower alkyl cyanide, chromatography being carriedout on a column material, especially an RP-18 material, which is chargedwith hydrocarbon chains containing 18 carbon atoms, and employing aneluant containing a nitrile, especially a lower alkylnitrile, inparticular acetonitrile, especially a mixture of nitrile/water, inparticular a mixture of acetonitrile/water, preferably in a ratio ofnitrile to water of ca. 1:99 to 99:1, primarily 1:9 to 9:1, e.g. between2:8 and 7:3, e.g. 3:7 or 4:6. This separation may follow on to afiltration with a molecular sieve, or is preferably effected directlyusing the residue after adsorption of the epothilones from the culturemedium containing a complex-forming component onto a resin, as describedabove (“variant 1”). One advantage of separation with an eluantcontaining a lower alkylcyanide over that using alcohols, such asmethanol, is the better separation of epothilones A and B.

The invention relates preferably to a process for the preparation ofepothilones, which

a) comprises a process for the concentration of epothilones, especiallyepothilone A and/or B, in particular epothilone B, in a culture mediumfor the biotechnological preparation of this (these) compound(s), whichcontains a microorganism which is suitable for this preparation,especially a Sorangium strain, especially of the type Sorangiumcellulosum Soce90, or a mutant arising therefrom, in particular thestrain having reference BCE 33/10, especially the strain havingreference BCE 63/114, water and other usual appropriate constituents ofculture media, whereby a cyclodextrin or a cyclodextrin derivative, or amixture of two or more of these compounds is added to the medium,especially one or more, preferably one or two or more cyclodextrinsselected from α-cyclodextrin (6), β-cyclodextrin (7), γ-cyclodextrin(8), δ-cyclodextrin (9), ε-cyclodextrin (10), ζ-cyclodextrin (11),η-cyclodextrin (12), and θ-cyclodextrin (13), especially α-cyclodextrin,β-cyclodextrin or γ-cyclodextrin; or primarily a cyclodextrin derivativeor mixture of cyclodextrin derivatives selected from derivatives of acyclodextrin, in which one or more up to all of the hydroxy groups areetherified to an alkyl ether, especially lower alkyl, such as methyl orethyl ether, also propyl or butyl ether; an aryl-hydroxyalkyl ether,such as phenyl-hydroxy-lower-alkyl, especially phenyl-hydroxyethylether; a hydroxyalkyl ether, in particular hydroxy-lower-alkyl ether,especially 2-hydroxyethyl, hydroxypropyl such as 2-hydroxypropyl orhydroxybutyl such as 2-hydroxybutyl ether; a carboxyalkyl ether, inparticular carboxy-lower-alkyl ether, especially carboxymethyl orcarboxyethyl ether; a derivatised carboxyalkyl ether, in particular aderivatised carboxy-lower-alkyl ether in which the derivatised carboxyis aminocarbonyl, mono- or di-lower-alkyl-aminocarbonyl, morpholino-,piperidino-, pyrrolidino- or piperazinocarbonyl, or alkyloxycarbonyl, inparticular lower alkoxycarbonyl, such as preferably a loweralkoxycarbonyl-lower-alkyl ether, for example methyloxycarbonylpropylether or ethyloxycarbonylpropyl ether; a sulfoalkyl ether, in particularsulfo-lower-alkyl ether, especially sulfobutyl ether; a cyclodextrin inwhich one or more OH groups are etherified with a radical of formula

—O-[alk-O—]_(n)—H

 wherein alk is alkyl, especially lower alkyl, and n is a whole numberfrom 2 to 12, especially 2 to 5, in particular 2 or 3; a cyclodextrin inwhich one or more OH groups are etherified with a radical of formula

 wherein R′ is hydrogen, hydroxy, —O-(alk-O)_(z)—H,—O-(Alk(-R)—O—)_(p)—H or —O-(alk(-R)—O—)_(q)-alk-CO—Y; alk in all casesis alkyl, especially lower alkyl; m, n, p, q and z are a whole numberfrom 1 to 12, preferably 1 to 5, in particular 1 to 3; and Y is OR₁ orNR₂R₃, wherein R₁, R₂ and R₃ independently of one another, are hydrogenor lower alkyl, or R₂ and R₃ combined together with the linking nitrogensignify morpholino, piperidino, pyrrolidino or piperazino;

or a branched cyclodextrin, in which etherifications or acetals withother sugars are present, and which are selected from glucosyl-,diglucosyl-(G₂-β-cyclodextrin), maltosyl- or di-maltosyl-cyclodextrin,or N-acetylglucosaminyl-, glucosaminyl-, N-acetylgalactosaminyl- andgalactosaminyl-cyclodextrin; or a lower alkanoyl, such as acetyl esterof a cyclodextrin; and

b) comprises a step for separating the epothilones, especiallyepothilones A and B, from one another, which is characterised bychromatography on a reversed-phase column with an eluant containing alower alkylcyanide, the chromatography being carried out on a columnmaterial, especially an RP-18 material, which is charged withhydrocarbon chains containing 18 carbon atoms, and employing an eluantcontaining a lower alkylnitrile, especially acetonitrile, in particulara mixture of lower alkylnitrile/water, preferably a mixture ofacetonitrile/water, preferably in a ratio of lower alkylnitrile to waterof ca. 1:99 to 99:1, primarily 1:9 to 9:1, e.g. between 2:8 and 7:3,e.g. 3:7 or 4:6, whereby if desired, it is possible to use further stepsfor working up and purification.

The invention also relates in particular to a mutant derived from thestrain Sorangium cellulosum Soce90, especially a strain of Sorangiumcellulosum which is obtainable by mutagenesis, preferably by one or moreUV-induced mutagenesis steps (in particular by UV radiation in the range200 to 400, especially 250 to 300 nm) with subsequent searching in eachstep for mutants having increased epothilone production (in particularincreased epothilone concentration in the culture medium), this strainunder otherwise identical conditions producing more epothilones, inparticular more epothilone A and/or B, especially epothilone B, thanSorangium cellulosum Soce90, especially the Sorangium cellulosum strainBCE 33/10, in particular BCE 114.

The invention relates in particular to the individual process stepsnamed in the examples or any combination thereof, the culture medianamed therein, crystal forms and the strain described therein.

The invention also relates to new crystal forms of epothilone B,especially a crystal form of epothilone B described as modification Band in particular described as modification A.

The crystal forms can be distinguished in particular by their X-raydiagrams. X-ray diagrams taken with a diffractometer and usingCu-Kα₁-radiation are preferably used to characterize solids of organiccompounds. X-ray diffraction diagrams are used particularly successfullyto determine the crystal modification of a substance. To characterizethe existing crystal modification A and crystal modification B ofepothilone B, the measurements are made at an angle range (2θ) of 2° and35° with samples of substance that are kept at room temperature.

The X-ray diffraction diagram thus determined (reflection lines andintensities of the most important lines) from crystal modification A(modification A) of epothilone B is characterized by the followingtable.

2θ Intensity  7.7 very strong 10.6 weak 13.6 average 14.4 average 15.5average 16.4 weak 16.8 weak 17.1 weak 17.3 weak 17.7 weak 18.5 weak 20.7strong 21.2 strong 21.9 weak 22.4 weak 23.3 strong 25.9 average 31.2weak 32.0 average

The invention also relates in particular to a new crystal form ofepothilones B, which is characterised by a melting point of more than120° C., especially between 120° C. and 128° C., in particular 124-125°C. Surprisingly, this value is considerably higher than the valuespreviously described in litreature. The invention relates especially toa crystal form of epothilone B, which is characterised by the X-raydiffraction diagram of the crystal form A and a melting point of above120° C., especially between 120° C. and 128° C., for example between124° C. and 125° C.

The X-ray diffraction diagram thus determined (reflection lines andintensities of the most important lines) of crystal modification B(modification B) of epothilone B is characterized by the followingtable.

2θ Intensity  6.9 very strong  8.0 weak  8.3 average 10.8 strong 11.5average 12.4 weak 13.1 strong 15.5 weak 16.2 weak 16.7 average 18.1average 18.6 average 20.4 weak 20.9 strong 21.3 weak 21.5 very weak 22.5average 24.2 weak 25.1 average

The invention also relates in particular to a new crystal form ofepothilones B, which is characterised by a melting point of more than120° C., especially between 124 and 125° C. Surprisingly, this value isconsiderably higher than the values previously described in litreature.

The new crystal forms are especially stable, particularly crystal formA, and they are therefore suitable as active ingredients for solid formsof administration, for storing in solid form or as intermediates (withparticularly good storability) in the preparation of solid or liquidforms of administration.

The invention also relates to the use of the new crystal forms,especially crystal form B, but primarily crystal form A (all referred tohereinafter as active ingredient) in the production of pharmaceuticalpreparations, new pharmaceutical preparations which contain these newcrystal forms, and/or the use of these compounds in the treatment ofproliferative diseases, such as tumours. In the following, wherepharmaceutical preparations or compositions which comprise or containthe active ingredient are mentioned, in the case of liquid compositionsor compositions which no longer contain the crystal form as such, thisis always understood to mean also the pharmaceutical preparationsobtainable using the crystal forms (for example infusion solutionsobtained using crystal forms A or B of epothilone B), even if they nolonger contain the respective crystal form (for example because theyexist in solution).

The invention also relates especially to the use of a new crystal formof epothilone B, especially the crystal form B or in particular crystalform A, in the production of pharmaceutical preparations, characterisedby mixing a new crystal form of epothilone B with one or more carriers.

The invention also relates to a method of treating warm-blooded animalssuffering from a proliferative disease, characterised by administering adose of epothilone B which is effective for treating said disease in oneor the new crystal forms to a warm-blooded animal requiring suchtreatment, also including in particular the treatment with thosepreparations that are produced using one of the new crystal forms.

To produce the pharmaceutical preparations, the active ingredient may beused for example in such a way that the pharmaceutical preparationscontain an effective amount of the active ingredient together or in amixture with a significant amount of one or more organic or inorganic,liquid or solid, pharmaceutically acceptable carriers.

The invention also relates to a pharmaceutical composition which issuitable for administration to a warm-blooded animal, especially humans,in the treatment of a proliferative disease, such as a tumour, thecomposition containing an amount of active ingredient that is suitablefor treating said disease, together with a pharmaceutically acceptablecarrier.

The pharmaceutical compositions according to the invention are thoseintended for enteral, especially nasal, rectal or oral, or preferablyparenteral, especially intramuscular or intravenous administration towarm-blooded animals, especially humans, and they contain an effectivedose of the active ingredient on its own or together with a significantamount of a pharmaceutically acceptable carrier. The dose of the activeingredient is dependent on the type of warm-blooded animal, the bodyweight, the age and the individual condition, individual pharmacokineticsituations, the disease to be treated and the type of administration.

The pharmaceutical compositions contain ca. 0.0001% to ca. 95%,preferably 0.001% to 10% or 20% to ca. 90% of active ingredient.Pharmaceutical compositions according to the invention may be presentfor example in unit dose forms, such as in the form of ampoules, vials,suppositories, dragées, tablets or capsules.

The pharmaceutical compositions according to the present invention areproduced by known processes, for example by conventional dissolving,lyophilizing, mixing, granulating or manufacturing processes.

Solutions of the active ingredient, also suspensions, and in particularaqueous solutions or suspensions, are preferably employed, whereby it isalso possible, for example in the case of lyophilised compositions whichcontain the active ingredient on its own or together with apharmaceutically acceptable carrier, for example mannitol, for thesolutions or suspensions to be prepared prior to administration. Thepharmaceutical compositions may be sterilised and/or may containexcipients, for example preservatives, stabilisers, moisture-retainingagents and/or emulsion-forming agents, dissolving aids, salts forregulating osmotic pressure and/or buffers, and they are produced byknown processes, for example by conventional dissolving or lyophilisingprocesses. The solutions or suspensions mentioned may compriseviscosity-increasing substances, such as sodium carboxymethylcellulose,carboxymethylcellulose, dextran, polyvinylpyrrolidone or gelatin.

Suspensions in oil contain as the oil component vegetable oils,synthetic oils or semi-synthetic oils, which are customary for injectionpurposes. Notable examples are in particular liquid fatty acid esters,which contain as the acid component a long-chained fatty acid with 8 to22, especially 12 to 22, carbon atoms, for example lauric acid,tridecylic acid, myristic acid, pentadecylic acid, palmitic acid,margaric acid, stearic acid, arachidic acid, behenic acid orcorresponding unsaturated acids, for example oleic acid, elaidic acid,erucic acid, brassidic acid or linoleic acid, if desired with theaddition of antioxidants, for example vitamin E, β-carotene or3,5-di-tert-butyl-4-hydroxytoluene. The alcoholic component of thesefatty acid esters preferably has a maximum of 6 carbon atoms and is amono- or polyhydroxy alcohol, for example a mono-, di- or tri-hydroxyalcohol, for example methanol, ethanol, propanol, butanol or pentanol,or an isomer thereof, but especially glycol and glycerol. The followingexamples of fatty acid esters may be mentioned in particular: propylmyristate, isopropyl palmitate, “Labrafil M 2375” (polyoxyethyleneglycerol trioleate, Gattefosse, Paris), “Miglyol 812” (triglyceride ofsaturated fatty acids having a chain length of 8 to 12 carbon atoms,Hüls AG, Germany), but in particular vegetable oils such as cottonseedoil, almond oil, olive oil, castor oil, sesame oil, soybean oil and inparticular peanut oil.

The injection or infusion preparations are produced according tocustomary methods under sterile conditions; the same applies also to thefilling of the compositions into ampoules or vials and sealedcontainers.

Preference is given to an infusion solution which contains the activeingredient and a pharmaceutically acceptable organic solvent.

The pharmaceutically acceptable organic solvents which may be used in aformulation according to the invention can be selected from all suchsolvents which are familiar to a person skilled in the art. The solventis preferably selected from an alcohol, e.g. absolute ethanol,ethanol/water mixtures, preferably 70% ethanol, polyethylene glycol 300,polyethylene glycol 400, polypropylene glycol and N-methylpyrrolidone,especially polypropylene glycol or 70% ethanol.

Particular preference is given to a formulation in pure polyethyleneglycol, which is diluted prior to infusion in an appropriate solution,such as physiological saline.

The active ingredient is present in the formulation in a concentrationof 0.001 to 100 mg/ml, preferably from ca. 0.05 to 5 mg/ml, or from 5 to50 mg/ml.

Formulations of this type are easily stored as vials or ampoules. Thevials or ampoules are typically made of glass, e.g. boron silicate. Thevials or ampoules may be appropriate for any volume which is known fromthe prior art. They are preferably of sufficient size to be able toaccept 0.5 to 5 ml of the formulation.

Prior to administration, the formulations have to be diluted in anaqueous medium suitable for intravenous administration before the activeingredient can be administered to patients.

It is preferable for the infusion solution to have the same or basicallythe same osmotic pressure as body fluids. Consequently, the aqueousmedium contains an isotonic agent which has the effect of rendering theosmotic pressure of the infusion solution the same or basically the sameas the osmotic pressure of body fluids.

The isotonic agent can be selected from all agents that are familiar toa person skilled in the art, for example mannitol, dextrose, glucose andsodium chloride. The isotonic agent is preferably glucose or sodiumchloride. The isotonic agents may be used in quantities which impart thesame or basically the same osmotic pressure to the infusion solution asbody fluids. The exact quantities required can be determined by routineexperiments and depend on the composition of the infusion solution andthe type of isotonic agent.

The concentration of isotonizing agent in the aqueous medium depends onthe type of each agent used. If glucose is used, it is preferably usedin a concentration of 1 to 5% w/v, preferably 5% w/v. If the isotonizingagent is sodium chloride, it is preferably used in quantities of up to1%, preferably ca. 0.9% w/v.

The infusion solution can be diluted with the aqueous medium. The amountof aqueous medium used is chosen according to the desired concentrationof active ingredient in the infusion solution. The infusion solution ispreferably produced by mixing a vial or an ampoule containing theinfusion concentrate (see above) with an aqueous medium, so that avolume of between 200 ml and 1000 ml is attained with the aqueousmedium. Infusion solutions may contain other additives that are normallyused in formulations for intravenous administration. These additivesalso include antioxidants.

Antioxidants may be used to protect the active ingredient fromdegradation by oxidation. Antioxidants may be selected from those whichare familiar to the person skilled in the art and which are suitable forintravenous formulations. The amount of antioxidant can be determined byroutine experiments. As an alternative to adding an antioxidant, oradditionally thereto, the antioxidant effect can be achieved byrestricting the oxygen (air) contact with the infusion solution. Thiscan be achieved in a simple way, by treating the vessel containing theinfusion solution with an inert gas, e.g. nitrogen or argon.

Infusion solutions can be produced by mixing an ampoule or a vial withthe aqueous medium, e.g. a 5% glucose solution in WFI in an appropriatecontainer, e.g. an infusion bag or an infusion bottle.

Containers for the infusion solutions may be selected from conventionalcontainers that are non-reactive with the infusion solution. Among thosesuitable are glass containers, especially of boron silicate, but plasticcontainers such as plastic infusion bags, are preferred.

Plastic containers may also be made of thermoplastic polymers. Theplastic materials may also contain additives, e.g. softeners, fillers,antioxidants, antistatic agents or other customary additives.

Suitable plastics for the present invention should be resistant toelevated temperatures used for sterilisation. Preferred plastic infusionbags are the PVC materials which are known to the person skilled in theart.

A large range of container sizes may be considered. When selecting thesize of the container, the factors to be taken into consideration areespecially the solubility of epothilones in an aqueous medium, easyhandling, and if appropriate, storage of the container. It is preferableto use containers which hold between ca. 200 and 1000 ml of infusionsolution.

Owing to their good formulating properties, the new crystal forms ofepothilone B according to the invention are especially suitable for thesimple and reproducible production of the said infusion solutions.However, the new crystal forms are especially suitable for theproduction of pharmaceutical formulations which contain the activeingredient in solid form, for example oral formulations.

Pharmaceutical formulations for oral application may be obtained bycombining the active ingredient with solid carriers, if desired bygranulating the resultant mixture, and further processing the mixture,if desired or if necessary, after adding suitable adjuvants, intotablets, dragee cores or capsules. It is also possible to embed them inplastic substrates which enable the active ingredient to be diffused orreleased in measured quantities.

Suitable pharmaceutically employable carriers are especially fillers,such as lactose, saccharose, mannitol or sorbitol, cellulosepreparations, and/or calcium phosphates, for example tricalciumphosphate or calcium hydrogen phosphate, and binders, such as starches,for example maize, wheat, rice or potato starch, gelatin, tragacanth,methyl cellulose, hydroxypropyl methyl cellulose, sodiumcarboxymethylcellulose, and/or polyvinyl pyrrolidone, and/or, ifdesired, disintegrators, such as the above-mentioned starches,crosslinked vinylpyrrolidones, agar, alginic acid or a salt thereof,such as sodium alginate. Adjuvants are in particular flow-improvingagents and lubricants, e.g. silicates, talcum, stearic acid or saltsthereof, such as magnesium or calcium stearate and/or polyethyleneglycol. Dragee cores are provided, if desired, with appropriategastric-juice-resistant coatings, using inter alia concentrated sugarsolutions, gum arabic, talcum, polyvinyl pyrrolidone, polyethyleneglycol and/or titanium dioxide, or coating solutions in suitable organicsolvents, or in order to produce gastric-juice-resistant coatings,solutions of appropriate cellulose preparations, such as ethyl cellulosephthalate or hydroxypropyl methyl cellulose phthalate. Capsules are drycapsules consisting of gelatin or pectin, and if required, a softenersuch as glycerol or sorbitol. The dry capsules may contain the activeingredient in the form of granules, for example with fillers, such aslactose, binders, such as starches, and/or lubricants, such as talc ormagnesium stearate, and where appropriate stabilizers. In soft capsules,the active ingredient may be present in dissolved or preferablysuspended form, whereby oily adjuvants such as fat oils, paraffin oil orliquid propylene glycols are added; stabilizers and/or antibacterialadditives may also be added. Dyes or pigments can be added to thetablets or dragee coatings, for example to improve identification or todistinguish different dosages of active ingredient.

The usage in the treatment of a proliferative disease with one of thecrystal forms B and in particular A preferably takes place whereby thecrystal form (preferably as for the usage in the preparation of aninfusion solution, as described above) is administered to a warm-bloodedanimal, especially a human, in a dose which can be determined at between20 and 133%, preferably between 25 and 100%, of the Maximum ToleratedDose (MTD) by standard methods, for example using a modified Fibronacciseries, in which the increases in dosages for successive amounts are100%, 67%, 50% and 40% followed by 33% for all subsequent doses; and, ifnecessary, one or more further doses administered in the dosage rangegiven above for the first dose, each dose after a period of time whichallows sufficient recovery of the individual being treated after thepreceding administration, in particular one week or more after the firstadministration, preferably 2 to 10 weeks, especially 3 to 6 weeks aftereach preceding administration. In general, this treatment scheme, inwhich a high dosage is administered once, twice or several times withsufficiently long intervals between the individual administrations forrecovery to take place, is preferred over a more frequent treatment withlower doses, since hospitalisation is less frequent and for a shorterperiod and an improved anti-tumour effect can be expected. The dosage ofepothilone B for humans is preferably between 0.1 and 50 mg/m²,preferably between 0.2 and 10 mg/m².

The following Examples serve to illustrate the invention withoutlimiting its scope.

Caution: When handling epothilones, appropriate protective measures mustbe taken, where necessary, in view of their high toxicity.

The 750 and 5000 litre fermenters used in the following are refinedsteel fermenters from the company Alpha AG, Nidau, Switzerland.

EXAMPLE 1 Preparation of the Strain BCE33/10 and the Strain BCE63/114 byMeans of Mutation and Selection

The strain employed is the mutant BCE33/10 (deposited at the GermanCollection of Microorganisms and Cell Cultures under number DSM 11999 onFeb. 9th, 1998) or the mutant BCE63/114 (deposited at the GermanCollection of Microorganisms and Cell Cultures under number DSM 12539 onNov. 27th, 1998), which is derived from the strain Sorangium cellulosumSoce90 by mutation and selection as described below. In liquid media,the mutant BCE33/10, as well as BCE63/114, forms bacilli typical ofSorangia, with rounded ends and a length of 3-6 μm, as well as a widthof ca. 1 μm. Sorangium cellulosum Soce90 was obtained from the GermanCollection of Microorganisms under number DSM 6773.

Preparation of the mutant BCE33/10 comprises three mutation steps withUV light and selections of individual colonies. The procedure in detailis carried out in accordance with the following operating steps

Cultivation from the ampoule: The cells of the DSM6773 ampoule aretransferred to 10 ml of G52 medium in a 50 ml Erlenmeyer flask andincubated for 6 days in an agitator at 30° C. and at 180 rpm. 5 ml ofthis culture are transferred to 50 ml of G52 medium (in a 200 mlErlenmeyer flask) and incubated at 180 rpm for 3 days in an agitator at30° C.

First UV mutation step and selection: Portions of 0.1 ml of the aboveculture are plated out onto several Petri dishes containing agar mediumS42. The plates are then each exposed to UV light (maximum radiationrange of 250-300 nm) for 90 or 120 seconds at 500 μwatt per cm². Theplates are then incubated for 7-9 days at 30° C., until individualcolonies of 1-2 mm are obtained. The cells of 100-150 colonies are theneach plated out from an individual colony by means of a plastic loop insectors onto Petri dishes containing S42 agar (4 sectors per plate) andincubated for 7 days at 30° C. The cells that have grown on an area ofca. 1 cm² agar surface are transferred by a plastic loop to 10 ml of G52medium in a 50 ml Erlenmeyer flask and incubated for 7 days at 180 rpmin an agitator at 30° C. 5 ml of this culture are transferred to 50 mlof G52 medium (in a 200 ml Erlenmeyer flask) and incubated at 180 rpmfor 3 days in an agitator at 30° C. 10 ml of this culture aretransferred to 50 ml of 23B3 medium and incubated for 7 days at 180 rpmin an agitator at 30° C.

To determine the amounts of epothilone A and epothilone B formed in thisculture, the following procedure is followed. The 50 ml culture solutionis filtered through a nylon sieve (150 μm pore size), and thepolystyrene resin Amberlite XAD16 retained on the sieve is rinsed with alittle water and subsequently added together with the filter to a 50 mlcentrifuge tube (Falcon Labware, Becton Dickinson AG Immengasse 7, 4056Basle). 10 ml of isopropanol (>99%) are added to the tube with thefilter. Afterwards, the well-sealed tube is shaken for 1 hour at 180 rpmin order dissolve the epothilone A and B, which is bonded to the resin,in the isopropanol. Subsequently, 1.5 ml of the liquid is centrifuged,and ca. 0.8 ml of the supernatant is added using a pipette to a HPLCtube. The HPLC analysis of these samples is effected as described belowunder HPLC analysis in the product analysis section. The HPLC analysisdetermines which culture contains the highest content of epothilone B.From the above-described sector plate of the corresponding colony (theplates have been stored at 4° C. in the meantime), cells from ca. 1 cm²of agar area are transferred by a plastic loop to 10 ml of G52 medium ina 50 ml Erlenmeyer flask and are incubated for 7 days at 180 rpm in anagitator at 30° C. 5 ml of this culture are transferred to 50 ml of G52medium (in a 200 ml Erlenmeyer flask) and incubated at 180 rpm for 3days in an agitator at 30° C.

Second and third UV mutation step and selection: The procedure isexactly the same as described above for the first UV mutation step,whereby the selected culture of the best colony from the first UVmutation is used for the second mutagenesis. For the third mutagenesis,the culture of the best colony from the second mutagenesis is usedaccordingly. The best colony after this third cycle of UV mutationsteps, followed by selection of the resulting strains for improvedepothilone B production, corresponds to mutant BCE33/10.

The strain BCE 63/114 is obtained from another (fourth) mutation stepfrom the strain BCE33/10, which is carried out in exactly the same wayas the above-mentioned mutation steps.

Preservation of the Strain

10 ml of a 3 day old culture in G52 medium (50 ml medium in a 200 mlErlenmeyer flask, 30° C. and 180 rpm) are transferred to 50 ml of 23B3medium (in a 200 ml Erlenmeyer flask) and incubated for 3 days at 180rpm in an agitator at 30° C. 1 ml portions of this culture are removedin a form which is as homogeneous as possible (prior to each removal theculture is shaken by hand in the Erlenmeyer flask) together with thepolystyrene resin Amberlite XAD16 (polystyrene adsorption resin, Rohm &Haas, Frankfurt, Germany), then filled into 1.8 ml Nunc cryotubes (A/SNunc, DK 4000 Roslide, Denmark) and stored either at −70° C. or inliquid nitrogen.

Cultivation of the strains from these ampoules is effected by heatingthem in the air to room temperature, and subsequently transferring theentire content of the cryotube to 10 ml G52 medium in an 50 mlErlenmeyer flask and incubating for 5-7 days at 180 rpm in an agitatorat 30° C.

Media

G52 Medium: yeast extract, low in salt (Springer, Maison Alfort, France)2 g/l MgSO₄ (7H₂O) 1 g/l CaCl₂ (2H₂O) 1 g/l soya meal defatted (MucedolaS.r.l., Settimo Milan, Italy) 2 g/l potato starch Noredux (Blattmann,Wädenswil, Switzerland) 8 g/l glucose anhydrous 2 g/l Fe-EDTA 8 g/l(Product No. 03625, Fluka Chemie AG, CH) 1 ml/l pH 7.4, corrected withKOH Sterilisation: 20 mins. 120° C.

S42 Agar-Medium: as described S. Jaoua et al. Plasmid 28, 157-165 (1992)23B3 Medium: glucose 2 g/l potato starch Noredux 20 g/l (Blattmann,Wädenswil, Switzerland) soya meal defatted 16 g/l (Mucedola S.r.l.,Settimo Milan, Italy) Fe-EDTA 0.008 g/l (Product No. 03625, Fluka,Buchs, Switzerland) HEPES Fluka, Buchs, Switzerland 5 g/l polystyreneresin XAD16 (Rohm and Haas) 2% v/v H₂O deionised correction of pH to 7.8with NaOH sterilisation for 20 mins. at 120° C. (HEPES =4-(2-hydroxyethyl)-piperazine-1-ethanesulfonic acid)

EXAMPLE 2 Cultivation in Order to Produce the Epothilones

Strain: Sorangium cellulosum Soce-90 BCE 33/10

EXAMPLE 1

Preservation of the Strain: In liquid N₂, as in Example 1.

Media: Precultures and intermediate cultures G52 Main culture 1B12 G52Medium yeast extract, low in salt 2 g/l (BioSpringer, Maison AlfortFrance) MgSO₄ (7H₂O) 1 g/l CaCl₂ (2H₂O) 1 g/l soya meal defattedSoyamine 50T 2 g/l (Lucas Meyer, Hamburg, Germany) potato starch NoreduxA-150 8 g/l (Blattmann, Waedenswil, Switzerland) glucose anhydrous 2 g/lEDTA-Fe(III)-Na salt (8 g/l) 1 ml/l pH 7.4, corrected with KOHSterilisation: 20 mins. 120° C.

1B12 medium: potato starch Noredux A-150 20 g/l (Blattmann, Waedenswil,Switzerland) soya meal defatted Soyamine 50T 11 g/l (Lucas Meyer,Hamburg, Germany) EDTA-Fe(III)-Na salt  8 mg/l pH 7.8, corrected withKOH Sterilisation: 20 mins. 120° C.

Addition of Cyclodextrins and Cyclodextrin Derivatives:

Cyclodextrins (Fluka, Buchs, Switzerland, or Wacker Chemie, Munich,Germany) in different concentrations are sterilised separately and addedto the 1B12 medium prior to seeding.

Cultivation: 1 ml of the suspension of Sorangium cellulosum Soce-90 BCE33/10 from a liquid N₂ ampoule is transferred to 10 ml of G52 medium (ina 50 ml Erlenmeyer flask) and incubated for 3 days at 180 rpm in anagitator at 30° C., 25 mm displacement. 5 ml of this culture is added to45 ml of G52 medium (in a 200 ml Erlenmeyer flask) and incubated for 3days at 180 rpm in an agitator at 30° C., 25 mm displacement. 50 ml ofthis culture is then added to 450 ml of G52 medium (in a 2 litreErlenmeyer flask) and incubated for 3 days at 180 rpm in an agitator at30° C., 50 mm displacement.

Maintenance culture: The culture is overseeded every 3-4 days, by adding50 ml of culture to 450 ml of G52 medium (in a 2 litre Erlenmeyerflask). All experiments and fermentations are carried out by startingwith this maintenance culture.

Tests in a Flask:

(I) Preculture in an Agitating Flask:

Starting with the 500 ml of maintenance culture, 1×450 ml of G52 mediumare seeded with 50 ml of the maintenance culture and incubated for 4days at 180 rpm in an agitator at 30° C., 50 mm displacement.

(ii) Main Culture in the Agitating Flask:

40 ml of 1B12 medium plus 5 g/l 4-morpholine-propane-sulfonic acid(=MOPS) powder (in a 200 ml Erlenmeyer flask) are mixed with 5 ml of a10× concentrated cyclodextrin solution, seeded with 10 ml of precultureand incubated for 5 days at 180 rpm in an agitator at 30° C., 50 mmdisplacement.

Fermentation: Fermentations are carried out on a scale of 10 litres, 100litres and 500 litres. 20 litre and 100 litre fermentations serve as anintermediate culture step. Whereas the precultures and intermediatecultures are seeded as the maintenance culture 10% (v/v), the maincultures are seeded with 20% (v/v) of the intermediate culture.Important: In contrast to the agitating cultures, the ingredients of themedia for the fermentation are calculated on the final culture volumeincluding the inoculum. If, for example, 18 litres of medium+2 litres ofinoculum are combined, then substances for 20 litres are weighed in, butare only mixed with 18 litres!

Preculture in an Agitating Flask:

Starting with the 500 ml maintenance culture, 4×450 ml of G52 medium (ina 2 litre Erlenmeyer flask) are each seeded with 50 ml thereof, andincubated for 4 days at 180 rpm in an agitator at 30° C., 50 mmdisplacement.

Intermediate Culture, 20 litres or 100 litres:

20 litres: 18 litres of G52 medium in a fermenter having a total volumeof 30 litres are seeded with 2 litres of the preculture. Cultivationlasts for 3-4 days, and the conditions are: 30° C., 250 rpm, 0.5 litresair per litre liquid per min, 0.5 bars excess pressure, no pH control.

100 litres: 90 litres of G52 medium in a fermenter having a total volumeof 150 litres are seeded with 10 litres of the 20 litre intermediateculture. Cultivation lasts for 3-4 days, and the conditions are: 30° C.,150 rpm, 0.5 litres of air per litre liquid per min, 0.5 bars excesspressure, no pH control.

Main Culture, 10 litres, 100 litres or 500 litres:

10 litres: The media substances for 10 litres of 1B12 medium aresterilised in 7 litres of water, then 1 litre of a sterile 10%2-(hydroxypropyl)-β-cyclodextrin solution are added, and seeded with 2litres of a 20 litre intermediate culture. The duration of the mainculture is 6-7 days, and the conditions are: 30° C., 250 rpm, 0.5 litresof air per litre of liquid per min, 0.5 bars excess pressure, pH controlwith H₂SO₄/KOH to pH 7.6+/−0.5 (i.e. no control between pH 7.1 and 8.1).

100 litres: The media substances for 100 litres of 1B12 medium aresterilised in 70 litres of water, then 10 litres of a sterile 10%2-(hydroxypropyl)-β-cyclodextrin solution are added, and seeded with 20litres of a 20 litre intermediate culture. The duration of the mainculture is 6-7 days, and the conditions are: 30° C., 200 rpm, 0.5 litresair per litre liquid per min., 0.5 bars excess pressure, pH control withH₂SO_(4/)KOH to pH 7.6+/−0.5. The chain of seeding for a 100 litrefermentation is shown schematically as follows:

500 litres: The media substances for 500 litres of 1B12 medium aresterilised in 350 litres of water, then 50 litres of a sterile 10%2-(hydroxypropyl)-β-cyclodextrin solution are added, and seeded with 100litres of a 100 litre intermediate culture. The duration of the mainculture is 6-7 days, and the conditions are: 30° C., 120 rpm, 0.5 litresair per litre liquid per min., 0.5 bars excess pressure, pH control withH₂SO₄/KOH to pH 7.6+/−0.5.

Product Analysis:

Preparation of the Sample:

50 ml samples are mixed with 2 ml of polystyrene resin Amberlite XAD16(Rohm+Haas, Frankfurt, Germany) and shaken at 180 rpm for one hour at30° C. The resin is subsequently filtered using a 150 μm nylon sieve,washed with a little water and then added together with the filter to a15 ml Nunc tube.

Elution of the Product from the Resin:

10 ml of isopropanol (>99%) are added to the tube with the filter andthe resin. Afterwards, the sealed tube is shaken for 30 minutes at roomtemperature on a Rota-Mixer (Labinco BV, Netherlands). Then, 2 ml of theliquid are centrifuged off and the supernatant is added using a pipetteto HPLC tubes.

HPLC Analysis:

Column Waters-Symetry C18, 100 × 4 mm, 3.5 μm WAT066220 + preliminarycolumn 3.9 × 20 mm WAT054225 Solvents A: 0.02% phosphoric acid B:Acetonitrile (HPLC-Quality) Gradient  41% B from 0 to 7 min. 100% B from7.2 to 7.8 min.  41%B from 8 to 12 min. Oven temp. 30° C. Detection 250nm, UV-DAD detection Injection vol. 10 μl Retention time Epo A: 4.30 minEpo B: 5.38 min

EXAMPLE 2A

Effect of the Addition of Cyclodextrin and Cyclodextrin Derivatives tothe Epothilone Concentrations Attained.

All the cyclodextrin derivatives tested here come from the companyFluka, Buchs, CH. The tests are carried out in 200 ml agitating flaskswith 50 ml culture volume. As controls, flasks with adsorber resinAmberlite XAD-16 (Rohm & Haas, Frankfurt, Germany) and without anyadsorber addition are used. After incubation for 5 days, the followingepothilone titres can be determined by HPLC:

TABLE 1 order Conc Epo A Epo B Addition No. [% w/v]¹ [mg/l] [mg/l]Amberlite XAD-16 (v/v) 2.0 9.2 3.8 (% v/v)2-hydroxypropyl-β-cyclodextrin 56332 0.1 2.7 1.72-hydroxypropyl-β-cyclodextrin 56332 0.5 4.7 3.32-hydroxypropyl-β-cyclodextrin 56332 1.0 4.7 3.42-hydroxypropyl-β-cyclodextrin 56332 2.0 4.7 4.12-hydroxypropyl-β-cyclodextrin 56332 5.0 1.7 0.52-hydroxypropyl-β-cyclodextrin 56330 0.5 1.2 1.22-hydroxypropyl-β-cyclodextrin 56330 1.0 1.2 1.22-hydroxypropyl-β-cyclodextrin 56330 5.0 2.5 2.3 β-cyclodextrin 287070.1 1.6 1.3 β-cyclodextrin 28707 0.5 3.6 2.5 β-cyclodextrin 28707 1.04.8 3.7 β-cyclodextrin 28707 2.0 4.8 2.9 β-cyclodextrin 28707 5.0 1.10.4 methyl-β-cyclodextrin 66292 0.5 0.8 <0.3 methyl-β-cyclodextrin 662921.0 <0.3 <0.3 methyl-β-cyclodextrin 66292 2.0 <0.3 <0.3 2,6di-o-methyl-β-cyclodextrin 39915 1.0 <0.3 <0.32-hydroxypropyl-γ-cyclodextrin 56334 0.1 0.3 <0.32-hydroxypropyl-γ-cyclodextrin 56334 0.5 0.9 0.82-hydroxypropyl-γ-cyclodextrin 56334 1.0 1.1 0.72-hydroxypropyl-γ-cyclodextrin 56334 2.0 2.6 0.72-hydroxypropyl-γ-cyclodextrin 56334 5.0 5.0 1.1 no addition 0.5 0.5¹Apart from Amberlite (% v/v), all percentages are by weight (% w/v).Few of the cyclodextrins tested (2,6-di-o-methyl-β-cyclodextrin,methyl-β-cyclodextrin) display no effect or a negative effect onepothiione production at the concentrations used. 1-2%2-hydroxy-propyl-β-cyclodextrin and β-cyclodextrin increase epothiloneproduction in the examples by 6 to 8 times compared with productionusing no cyclodextrins.

EXAMPLE 2B

10 litre Fermentation with 1% 2-(hydroxypropyl)-β-cyclodextrin):

Fermentation is carried out in a 15 litre glass fermenter. The mediumcontains 10 g/l of 2-(hydroxypropyl)-β-cyclodextrin from Wacker Chemie,Munich, Germany. The progress of fermentation is illustrated in Table 2.Fermentation is ended after 6 days and working up takes place.

TABLE 2 Progress of a 10 liter fermentation duration of culture Epo AEpo B [d] [mg/l] [mg/l] 0 0 0 1 0 0 2 0.5 0.3 3 1.8 2.5 4 3.0 5.1 5 3.75.9 6 3.6 5.7

Example 2C

100 litre Fermentation with 1% 2-(hydroxypropyl)-β-cyclodextrin):

Fermentation is carried out in a 50 litre fermenter. The medium contains10 g/l of 2-(hydroxypropyl)-β-cyclodextrin. The progress of fermentationis illustrated in Table 3. The fermentation is harvested after 7 daysand worked up.

TABLE 3 Progress of a 100 liter fermentation duration of culture Epo AEpo B [d] [mg/l] [mg/l] 0 0 0 1 0 0 2 0.3 0 3 0.9 1.1 4 1.5 2.3 5 1.63.3 6 1.8 3.7 7 1.8 3.5

EXAMPLE 2D

500 litre Fermentation with 1% 2-(hydroxypropyl)-β-cyclodextrin):

Fermentation is carried out in a 750 litre fermenter. The mediumcontains 10 g/l of 2-(hydroxypropyl)-β-cyclodextrin. The progress offermentation is illustrated in Table 4. The fermentation is harvestedafter 7 days and worked up.

TABLE 4 Progress of a 500 liter fermentation duration of culture Epo AEpo B [d] [mg/l] [mg/l] 1 0 0 2 0 0 3 0.6 0.6 4 1.7 2.2 5 3.1 4.5 6 3.15.1

EXAMPLE 2E

Comparison Example 10 litre Fermentation without Adding an Adsorber:

Fermentation is carried out in a 15 litre glass fermenter. The mediumdoes not contain any cyclodextrin or other adsorber. The progress offermentation is illustrated in Table 5. The fermentation is notharvested and worked up.

TABLE 5 Progress of a 10 liter fermentation without adsorber. durationof culture Epo A Epo B [d] [mg/l] [mg/l] 0 0 0 1 0 0 2 0 0 3 0 0 4 0.70.7 5 0.7 1.0 6 0.8 1.3

EXAMPLE 3 Working Up of the Epothilones: Isolation from a 500 litre MainCulture

The volume of harvest from the 500 litre main culture of example 2D is450 litres and is separated using a Westfalia clarifying separator TypeSA-20-06 (rpm=6500) into the liquid phase (centrifugate+rinsingwater=650 litres) and solid phase (cells=ca. 15 kg). The main part ofthe epothilones are found in the centrifugate. The centrifuged cell pulpcontains <15% of the determined epothilone portion and is not furtherprocessed. The 650 litre centrifugate is then placed in a 400 litrestirring vessel, mixed with 10 litres of Amberlite XAD-16(centrifugate:resin volume=65:1) and stirred. After a period of contactof ca. 2 hours, the resin is centrifugate away in a Heine overflowcentrifuge (basket content 40 litres; rpm=2800). The resin is dischargedfrom the centrifuge and washed with 10-15 litres of deionised water.Desorption is effected by stirring the resin twice, each time inportions with 30 litre of isopropanol in 30 litre glass stirring vesselsfor 30 minutes. Separation of the isopropanol phase from the resin takesplace using a suction filter. The isopropanol is then removed from thecombined isopropanol phases by adding 15-20 litres of water in avacuum-operated circulating evaporator (Schmid-Verdampfer) and theresulting water phase of ca. 10 litres is extracted 3× each time with 10litres of ethyl acetate. Extraction is effected in 30 litre glassstirring vessels. The ethyl acetate extract is concentrated to 3-5 litrein a vacuum-operated circulating evaporator (Schmid-Verdampfer) andafterwards concentrated to dryness in a rotary evaporator (Buchi type)under vacuum. The result is an ethyl acetate extract of 50.2 g. Theethyl acetate extract is dissolved in 500 ml of methanol, the insolubleportion filtered off using a folded filter, and the solution added to a10 kg Sephadex LH 20 column (Pharmacia Uppsala, Sweden) (column diameter20 cm, filling level ca. 1.2 m. Elution is effected with methanol aseluant. Epothilone A and B is present predominantly in fractions 21-23(at a fraction size of 1 litre). These fractions are concentrated todryness in a vacuum on a rotary evaporator (total weight 9.0 g). TheseSephadex peak fractions (9.0 g) are thereafter dissolved in 92 ml ofacetonitrile:water:methylene chloride=50:40:2, the solution filteredthrough a folded filter and added to a RP column (equipment Prepbar 200,Merck; 2.0 kg LiChrospher RP-18 Merck, grain size 12 μm, column diameter10 cm, filling level 42 cm; Merck, Darmstadt, Germany). Elution iseffected with acetonitrile:water=3:7 (flow rate=500 ml/min.; retentiontime of epothilone A=ca. 51-59 mins.; retention time of epothilone B=ca.60-69 mins.). Fractionation is monitored with a UV detector at 250 nm.The fractions are concentrated to dryness under vacuum on aBüchi-Rotavapor rotary evaporator. The weight of the epothilone A peakfraction is 700 mg, and according to HPLC (external standard) it has acontent of 75.1%. That of the epothilone B peak fraction is 1980 mg, andthe content according to HPLC (external standard) is 86.6%. Finally, theepothilone A fraction (700 mg) is crystallised from 5 ml of ethylacetate:toluene=2:3, and yields 170 mg of epothilone A purecrystallisate [content according to HLPC (% of area)=94.3%].Crystallisation of the epothilone B fraction (1980 mg) is effected from18 ml of methanol and yields 1440 mg of epothilone B pure crystallisate[content according to HPLC (%) of area=99.2%]. m.p. (Epothilone B):124-125° C.; ¹H-NMR data for Epothilone B: 500 MHz-NMR, solvent:DMSO-d6. Chemical displacement δ in ppm relative to TMS. s=singlet;d=doublet; m=multiplet

integral δ (Multiplicity) (number of H) 7.34 (s) 1 6.50 (s) 1 5.28 (d) 15.08 (d) 1 4.46 (d) 1 4.08 (m) 1 3.47 (m) 1 3.11 (m) 1 2.83 (dd) 1 2.64(s) 3 2.36 (m) 2 2.09 (s) 3 2.04 (m) 1 1.83 (m) 1 1.61 (m) 1 1.47-1.24(m) 4 1.18 (s) 6 1.13 (m) 2 1.06 (d) 3 0.89 (d + s, overlapping) 6 Σ =41

In this example (Example 3), epothilone B is obtained in the crystalmodification A, which is characterised by the X-ray diffraction diagramof modification A (see general part of the present disclosure).

EXAMPLE 4 Crystal Modification B of Epothilone B

50 mg of epothilone B (obtained for example as above) are suspended in 1ml of isopropanol and shaken for 24 hours at 25° C. The product isfiltered and dried. After drying under a high vacuum, epothilones B areobtained in the form of white crystals. The crystal modification of theproduct is characterised by the X-ray diffraction diagram ofmodification B (see general part of the present disclosure).

EXAMPLE 5 3000 litre Fermentation with 2-(hydroxypropyl)-β-cyclodextrin)

Fermentation is carried out with the strain BCE 63/114 in a 5000 litrefermenter in 1B12 medium (filled volume 3000 litres).

Maintenance culture: Preparation is effected as described in Example 1(strain preservation) and 2 for the strain BCE.33/10, but using insteadthe strain BCE63/114.

Precultures:

Preparation of the precultures is effected analogously to Example 2(ii), but with the following precultures and with strain BCE 63/114:

From a 500 ml maintenance culture [as described in example 2 (ii)], 50ml portions are placed in 4 Erlenmeyer flasks, thus producing four 500ml precultures in G52 medium for 3 days at 30° C. and at 180 rpm. These4 precultures (2 litres) are then used for three intermediate cultureseach of 20 litres (G52 medium, 4 days, 30° C., 250 rpm). 5 litreportions of these intermediate cultures are used to produce three 50litre intermediate cultures (G52 medium, 3 days, 30° C., 200 rpm). 50litres of these 50 litre intermediate cultures are used twice to growtwo 600 litre intermediate cultures (G52 medium, 4 days, 30° C., 120rpm).

Main culture: The media substances for 3000 litres are sterilised in2100 litres of water, then 300 litres of a sterile 10%2-(hydroxypropyl)-β-cyclodextrin solution are added, and seeded with 600litres of an intermediate culture. The duration of the main culture is6-7 days, and the conditions are: 30° C., 100 rpm, 0.5 litres of air perlitre liquid per minute, 0.5 bars excess pressure, pH control withH₂SO₄/KOH to pH 7.6+/−0.5 (i.e. no control between pH 7.1 and 8.1). Theprogress of fermentation is illustrated in Table 6.

TABLE 6 Progress of a 3000 liter fermentation duration of cultureEpothilone A Epothilone B [d] [mg/l] [mg/l] 0 0 0 1 0 0 2 0 0 3 2.1 1.64 4.1 2.9 5 5.2 3.8 6 5.5 4.3

Working Up and Isolation of the Epothilones from a 3000 litre MainCulture:

(I) Resin Binding, Desorption and Extraction of the Epothilones (ethylacetate extract)

The volume of harvest from the 3000 litre main culture is 2900 litresand is separated using a Westfalia clarifying separator Type SA-20-06(rpm=6500, flow rate 1400 litres/hour) into the liquid phase(centrifugate+rinsing water=2750 litres) and solid phase (cells=ca. 260kg). The main part of the epothilones are found in the centrifugate, Thecentrifuged cell pulp contain <15% of the determined epothilone portionand is not further processed. The 2750 litre centrifugate is then placedin a 4000 litre steel stirring vessel, mixed with 60 litres of AmberliteXAD-16 (centrifugate:resin volume=46:1) and stirred. After a period ofcontact of 16-20 hours, the resin is centrifuged away in a Heineoverflow centrifuge (basket content 40 litres; rpm=2800). The centrifugeis emptied by rinsing the basket content with deionised water when thecentrifuge is stationary. The XAD-16/deionised water slurry isthereafter freed from water on a suction filter (Ø 50 cm) and the resinwashed with 30 litres of deionised water. Desorption of the resin iseffected by stirring it in a 1600 litre stirring vessel twice, each timewith 220 litres of isopropanol for 30 minutes. Separation of theisopropanol phase from the resin takes place using a suction filter (Ø50 cm). The isopropanol is then removed from the isopropanol phase byadding 240-260 litres of water in a vacuum-operated circulatingevaporator (Schmid-Verdampfer) and the resulting water phase of ca. 125litre is extracted 3× each time with 100-125 litres of ethyl acetate.Extraction is effected in 1600 litre steel stirring vessels. The ethylacetate extracts are combined, concentrated to 3-5 litres invacuum-operated circulating evaporators (BüchiVerdampfer/Schmid-Verdampfer) and afterwards concentrated to dryness ina rotary evaporator (Büchi type) under vacuum. An ethyl acetate extractof 590 g is obtained.

HPLC Purification of the Ethyl Acetate Extract (separation ofepothilones A and B)

Ca. 300 g of the above-mentioned ethyl acetate extract (with a contentof ca. 1-1.5% epothilone B) are suspended in 1.5 litres ofacetonitrile/water=3/1 (v/v), the solution is filtered through a foldedfilter and the filtrate added to a C-18 RP column [Prochrom apparatuswith 30 cm internal column diameter (Prochrom, Champigneulles, France)25 kg YMC gel, ODS-A, 120 Angstroem pore diameter, 5-15 μm grain size,spherical]. Elution is effected with acetonitrile/water=4/6 (v/v) at aflow rate of 2300 ml/min. Fractionation is monitored by means of on-lineHPLC [rapid HPLC at high temperatures (ca. 80° C.), on a shortseparating column (4.6 mm internal diameter×75 mm length) and very smallRP-18 particles (3.5 μm spherical), typical analysis times are <1minute, detection at 250 nm]. The valuable fractions (those with onlyepothilone B) are combined, the acetonitrile removed by distillation andthe aqueous phase extracted twice with isopropyl acetate. The organicphases are concentrated by distillation and the isopropyl acetateextract is obtained as the residue of evaporation.

(iii) Silica Gel Filtration of the Isopropyl Acetate Extract

Ca. 10 g of the combined residues of evaporation thus obtained (with acontent of ca. 23% of epothilone B) are dissolved at room temperature in360 ml of ethyl acetate, the solution is filtered through a foldedfilter and added to a column of silica gel (Prochrom apparatus with 10cm internal column diameter, 1.5 kg ICN 18-32 μm). Elution is effectedwith ethyl acetate/n-hexane=4/1 (v/v) at a flow rate of 250 ml/min atroom temperature. Fractionation is monitored with a UV detector at 250nm. The valuable fractions are combined, the solvent removed bydistillation and concentrated to dryness.

(iv) Extraction of the Pure Epothilones

Ca. 48 g of the combined residues obtained under (iii) (with a contentof ca. 90% of epothilone B) are dissolved in 1150 ml of methanol, mixedwith 14.5 g of activated carbon and subsequently filtered through afolded filter. The clear filtrate is subsequently concentrated todryness and the residue is recrystallised from 317 ml of methanol. 29.5g of epothilone B are obtained in a purity of 99.7%, and with a meltingpoint of 124° C.

EXAMPLE 6 Infusion Concentrate

By dissolving in polyethylene glycol PEG 300, crystal modification A ofepothilone B, or crystal modification B of epothilone B, is produced ina preconcentrate to produce infusion solutions, and stored in vials.

What is claimed is:
 1. Crystalline epothilone B characterized by amelting point greater than 120° C. characterized by peaks in an x-raydiffraction pattern obtained with CuK radiation at about 7.7, 20.7, 21.2and 23.3 degrees 2 theta.
 2. Crystalline epothilone B according to claim1 characterized by peaks in an x-ray diffraction pattern obtained withCu-Kα₁ radiation at about 7.7, 13.6, 14.4, 15.5, 20.7, 21.2, 23.3, 25.9and 32.0 degrees 2 theta.
 3. Crystalline epothilone B according to claim1 having a melting point between 120° C. and 128° C.
 4. Crystallineepothilone B according to claim 3 having a melting point in the rangefrom 124° C.-125° C.
 5. Crystalline epothilone B characterized by amelting point greater than 120° C. characterized by peaks in an x-raydiffraction pattern obtained with Cu-Kα₁ radiation at about 6.9, 10.8,13.1 and 20.9 degrees 2 theta.
 6. Crystalline epothilone B according toclaim 5 characterized by peaks in an x-ray diffraction pattern obtainedwith Cu-Kα₁ radiation at about 6.9, 8.3, 10.8, 11.5, 13.1, 16.7, 18.1,18.6, 20.9, 22.5 and 25.1 degrees 2 theta.
 7. Crystalline epothilone Baccording to claim 5 having a melting point in the range from 124°C.-125° C.
 8. A method of preparing a pharmaceutical preparation, whichcomprises mixing crystalline epothilone B according to claim 1 with apharmaceutically acceptable carrier.
 9. A method of preparing apharmaceutical preparation, which comprises mixing crystallineepothilone B according to claim 5 with a pharmaceutically acceptablecarrier.
 10. Crystalline epothilone B of claim 1 having a melting pointin the range from 120° C.-128° C.
 11. Crystalline epothilone B of claim10 having a melting point in the range from 124° C. to 125° C.
 12. Thecrystalline epothilone B according to claim 5 which is characterized byan x-ray diffraction pattern obtained using Cu-Kα as follows: 2θIntensity  6.9 very strong  8.0 weak  8.3 average 10.8 strong 11.5average 12.4 weak 13.1 strong 15.5 weak 16.2 weak 16.7 average 18.1average 18.6 average 20.4 weak 20.9 strong 21.3 weak 21.5 very weak 22.5average 24.2 weak 25.1 average.


13. The crystal form of epothilone B according to claim 1, which ischaracterized by the X-ray diffraction diagram reproduced in the form ofa table, obtained using a diffractometer with Cu-Kα₁ radiation: 2θIntensity 7.7 Very strong 10.6 weak 13.6 Average 14.4 Average 15.5Average 16.4 weak 16.8 weak 17.1 weak 17.3 weak 17.7 weak 18.5 weak 20.7strong 21.2 strong 21.9 weak 22.4 weak 23.3 strong 25.9 Average 31.2weak 32.0 Average.


14. Method of treating a warm-blooded animal suffering from aproliferative disease, comprising administering a dosage of a crystalform of epothilone B as claimed in claim 1 or claim 5 which is effectivefor treating said disease to a warm-blooded animal requiring suchtreatment.